Our long-term objective is to understand the molecular mechanism of nucleotide excision repair (NER) in eukaryotes. DNA damage can be induced by radiation and many chemical carcinogens, and is potentially mutagenic and carcinogenic. NER is an important repair mechanism for removing a wide spectrum of different damage. Compromising NER activity would lead to a high risk of human carcinogenesis, such is the case of the cancer- prone human disease xeroderma pigmentosum (XP). We have been studying the NER mechanism by using the yeast Saccharomyces cerevisiae as a eukaryotic model organism. Yeast NER involves at least the following proteins and protein factors: Rad1, Rad2, Rad4, Rad7, Rad10, Rad14, Rad16, Rad23, Mms19, TFIIH, RPA, PCNA, RFC, DNA polymerases delta and epsilon, and DNA ligase I. NER pathway can be divided into five steps: damage recognition, incision, excision, repair synthesis, and DNA ligation. The roles of many proteins in NER and several steps of this repair pathway have been elucidated in eukaryotes. In contrast, the damage recognition step and roles of Rad23 and Mms19 proteins in NER have not been clearly defined. These important aspects of NER will be studied in this proposal. Our specific aims are (1) to delineate the damage recognition step of NER; (2) to elucidate the function of Rad23 protein in NER; and (3) to determine the role of MMS19 gene product in NER. The damage recognition step of NER will be analyzed with respect to protein binding to damaged DNA, correlation between damage binding and repair efficiency, and the ability of the damage binding factor to subsequently recruit other repair proteins for further assembly of the NER machinery. In vitro biochemical experiments and in vivo genetic experiments will be used to test several hypotheses regarding functions of Rad23 and Mms19 proteins in NER. These studies should lead to a better understanding of the NER pathway in eukaryotes, which in turn should advance our understanding on the interaction between human health and many genotoxic agents in our environment.